What is the role of non-native intermediates of beta-lactoglobulin in protein folding?
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The mechanism of alpha-->beta transition in folding of beta-lactoglobulin is discussed based on free energy landscape analysis of a long lattice model. It is found that helical propensity of beta-lactoglobulin is driven by conformational entropy and is intrinsically coded in its native structure. We propose a view on a role of folding intermediate, which is "on-pathway" but rich in non-native structures. The present results suggest that the native structure topology plays an important role in alpha-->beta transition. (+info)
Crystal structures of bovine beta-lactoglobulin in the orthorhombic space group C222(1). Structural differences between genetic variants A and B and features of the Tanford transition.
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The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue. (+info)
T cells of multiple sclerosis patients target a common environmental peptide that causes encephalitis in mice.
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Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA(193), which was targeted by most MS but not diabetes patients. BSA(193) was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA(193), in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry. (+info)
The methanol-induced conformational transitions of beta-lactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry.
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The methanol-induced conformational transitions under acidic conditions for beta-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with beta-sheets, alpha-helices, and both alpha-helices and beta-sheets, respectively, are studied under equilibrium conditions by electrospray ionization mass spectrometry (ESI-MS). The folding states of proteins in solution are monitored by the charge state distributions that they produce during ESI and by hydrogen/deuterium (H/D) exchange followed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induced alpha-helix structure. Intermediate states formed at about 35% methanol concentration are found to give bimodal charge state distributions. The same rate of H/D exchange is shown by the two contributions to the bimodal distributions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchange of proteins followed by ESI-MS shows that helical denatured states, populated at around 50% methanol concentration, transform into more protected structures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins. (+info)
Isolation and characterization of four bactericidal domains in the bovine beta-lactoglobulin.
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Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function. (+info)
Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin.
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The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG. (+info)
The specificity of monoglyceride-protein interactions and mechanism of the protein induced L(beta) to coagel phase transition.
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This study aims at gaining insight into the specificity and molecular mechanism of monoglyceride-protein interactions. We used beta-lactoglobulin (beta-LG) and lysozyme as model proteins and both monostearoylglycerol and monopalmitoylglycerol as defined gel phase monoglycerides. The monoglycerides were used in different combinations with the two negatively charged amphiphiles dicetylphosphate and distearylphosphate. The interactions were characterized using the monolayer technique, isothermal titration calorimetry, (2)H-nuclear magnetic resonance (NMR) using deuterium labelled monoglycerides and freeze fracture electron microscopy (EM). Our results show that lysozyme inserts efficiently into all monolayers tested, including pure monoglyceride layers. The insertion of beta-LG depends on the lipid composition of the monolayer and is promoted when the acylchains of the negatively charged amphiphile are shorter than that of the monoglyceride. The binding parameters found for the interaction of beta-LG and lysozyme with monoglyceride bilayers were generally similar. Moreover, in all cases a large exothermic binding enthalpy was observed which was found to depend on the nature of the monoglycerides but not of the proteins. (2)H-NMR and freeze fracture EM showed that this large enthalpy results from a protein mediated catalysis of the monoglyceride L(beta) to coagel phase transition. The mechanism of this phase transition consists of two steps, an initial protein mediated vesicle aggregation step which is followed by stacking and probably fusion of the bilayers. (+info)
Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris.
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Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions. (+info)